Human growth hormone (hGH) is a 22 kDa molecular weight single chain polypeptide, (hGH 22K), composed of 191 amino acids with two intra-chain disulfide bonds, produced by the anterior pituitary gland (1,2). However, circulating hGH is a complex mixture of different molecular forms, some of which are pituitary-derived, such as hGH 22K and hGH 20K, a human growth hormone 20 kDa molecular weight single chain polypeptide, while others are secreted by the placenta during pregnancy (hGH-V). Furthermore, other tropic hormones, placental lactogen (hPL) and prolactin (PRL), show significant sequence identity with hGH. Other hGH molecular variants derived from post-translational modifications such as deamidation, acylation, glycosylation and oligomerization (3) have also been described. Human growth hormone is coded for by two genes, hGH-N and hGH-V, which are clustered on chromosome 17 together with the highly homologous placental lactogen (hPL) gene (4,5). The main product of the pituitary-expressed hGH-N gene is the 191-amino-acid 22K hGH.
A secondary product of this gene is 20K hGH, derived by alternative mRNA splicing, which lacks 15 residues in the polypeptide chain, from amino acids 32 to 46 (6, 7). This hGH 20K represents 5-10% of pituitary hGH (3), and its biological properties have yet to be defined. While it certainly shares some functions of the 22K isoform, evidence also shows specific activities. Thus, hGH 20K does not bind to hGH receptors in human liver (8) or at least shows decreased binding (9) and has less insulin-like promoting activity (10). The 20K isoform competes with hGH for binding to rabbit mammary gland receptors, indicating that its effect is more like lactogen than somatogen, even though it promotes growth in the hypophysectomized rat (11) see also, for example, European Patent Application EP 587427.
hGH 20K is excreted at a slower rate than hGH and this prolonged persistence of hGH 20K in the circulation may contribute to its higher than expected bioactivity in vivo (Baumann et al, Endocrinology, Vol 117, No 4, 1309-13, 1985).
EP 587 427 disclosed a process for producing hGH 20K by a recombinant method.
Despite the clinical relevance of this peptide hormone family, there is little information on the concentrations of circulating isoforms or the relative contribution of each molecular form of the complex mixture. Selective assays to define hGH 22K and hGH 20K concentrations would be valuable both for diagnostics and basic research (12, 13, 14,). Tools such as monoclonal antibodies (mAb) which specifically block the effect of these proteins could be of great interest in helping to understand their specific biological actions. It has been suggested that the amount and relation between hGH 22K and hGH 20K in circulation could be of importance for special diseases and states of illness, such as diabetes, acromegaly, chronic liver and/or renal disease. A specific and precise method for measuring 20 kDa is thus highly needed.
No method, based on the use of a hGH 20K specific mAb, for specific detection and quantification exists today. Attempts have been done to raise hGH 20K specific mAbs with the purpose of developing immunoassays for hGH 20K, but without success. Reference is made to F Gomez et al, J of Immunoassay, 5 (364), 145-57 (1984). On page 155 of Gomez et al. it is stated "Since the precise pathophysiological relevance of 20K GH is still largely unknown, we felt appropriate to develop an immunoassay for it, obtaining first monoclonal anti hGH 20K antibodies with high specificity. Nevertheless, no stable hybridoma secreting specific anti 20K antibodies could be obtained despite selective immunization of the animals with a highly purified preparation of the variant".
For a long time there has thus been a need for a hGH 20K antibodies with high specificity which could be used in a specific and precise method for measuring hGH 20K. Such an antibody could possibly also be used for therapeutic applications when blocking the biological activity of hGH 20K.
We have now found a solution to the need of the detection and quantification of hGH 20K, as we have generated a mAb specific for hGH 20K. This mAb has been used successfully for the specific detection of this hormone in different types of assays and has also been found to specifically block its biological activity. For that reason it is also useful to study the biological activity of this hormone and analyze its biological significance.
In specific examples we describe the generation and characterization of this mAb. As comparitive antibodies, we also describe monoclonal antibodies specific for hGH 22K and one which recognizes both hGH 20K and hGH 22K.